In comparison to our detailed knowledge of the regulatory mechanisms governing the expression of gene clusters, such as the globin loci in hematopoietic cells, much less is known about the tissue specific expression of individual genes in other differentiated cell types. The genetic elements that confer temporal and spatial regulation on the expression of many genes lie outside the gene promoters and are buried deep within intronic or intergenic sequences. Moreover, there are functional barriers to prevent regulatory signals from one gene interfering with the expression of flanking loci that show divergent tissue-specificity. One example of a gene that is essential to the function of specialized epithelial cells and shows a complex expression pattern involving intronic regulatory elements, is the cystic fibrosis transmembrane conductance regulator gene (CFTR). Absence of the CFTR chloride ion channel impairs the function of secretory epithelia, causing cystic fibrosis (CF). The basal promoter of CFTR alone does not confer accurate temporal and spatial expression on the gene and little is known about the function of other elements regulating its expression. In this project we will investigate novel mechanisms controlling tissue-specific regulation of CFTR gene expression in the airway epithelium. We will identify and characterize cis-acting regulatory elements and transacting factors that determine airway-specific expression of CFTR. Moreover, we will establish the mechanisms of action of these control sequences within chromatin and determine how they influence gene expression. These experiments are likely to reveal regulatory pathways that are of general importance in gene expression in the airway epithelium. We will test the hypothesis that epithelial-specific regulation of CFTR gene expression is achieved by different mechanisms in the respiratory and intestinal epithelium. We propose that in both tissues, CFTR expression is controlled by transcription factors binding to intronic elements but that different trans-acting factors and cis-elements are utilized in the two epithelia. We further propose that CFTR is maintained in a chromatin domain, flanked by 5' and 3' boundary elements that facilitate coordinated regulation of gene expression in different cellular environments. Specific Aim 1 will define the tissue-specific cis-regulatory elements that orchestrate CFTR expression in the airway epithelium and identify the trans-acting factors that interact with these elements. Specific Aim 2 will determine the chromatin domain of the CFTR locus in airway epithelial cells and identify the factors that are involved in the maintenance of this domain in active or inactive configurations. These studies will address an important gap in our understanding of CFTR and provide opportunities to design novel CF therapies that modulate CFTR expression levels in vivo. Moreover, a better knowledge of CFTR regulation has the potential to increase the clinical success of gene therapy protocols. PUBLIC HEALTH RELEVANCE. The cystic fibrosis transmembrane conductance regulator gene (CFTR), that when mutated causes CF, has a complex pattern of tissue-specific and temporal regulation. The elements that control this are, in general, poorly characterized. Our team has made substantial progress on identifying and elucidating the control mechanisms for CFTR. The current research program builds upon this progress and concentrates on elucidating novel regulatory mechanisms that may be particularly relevant to CFTR expression in the airway. Translational opportunities that may arise from this work include modulating CFTR expression in vivo and the construction of efficient, tissue-specific gene therapy vectors.